Introduction

Hypercapnia during breath holding is believed to be the dominant driver behind the modulation of cerebral blood flow (CBF). Instead of being considered only as a hypercapnic challenge, breath hold can also be recognized as a form of brief hypoxia with concurrent mild hypercapnia.^1-3 We hypothesized that hypoxia and hypercapnia work synergistically to enhance CBF response to breath hold. Here we showed that the cerebrovascular responses to brief breath hold epochs were coupled not only with increased partial pressure of carbon dioxide (PCO₂), but also with a decrease in partial pressure of oxygen (PO₂). In spontaneous breathing, blood gas levels of O₂ and CO₂ are optimized by the feedback control of ventilation via chemoreflexes⁴ to regulate blood flow and O₂ delivery to the brain as part of a vital homeostatic process.⁵ The same process from chemosensing to CBF change is assumed to take place during breath hold as well. Increasing evidence showed that mild hypercapnia could increase the sensitivity of the CBF response to a very mild level of hypoxia and the ranges of mild PO₂ and PCO₂ changes reported are achievable by breath hold. We therefore examined breath hold in the framework of hemodynamic responses to mild hypoxia together with hypercapnia in humans.

Subjects and Methods

Participants: Ten healthy volunteers aged 22-48 years participated in both breath hold and exogenous CO₂ MRI. Methods: MRI was performed at 3-Tesla (Siemens Medical Germany) in Athinoula A. Martinos Center at Massachusetts General Hospital. Experimental procedures were explained to the subjects, and signed informed consent was obtained prior to participation in the study. Whole brain MRI datasets were acquired for each subject: 1) T1-weighted 3D-MEMPRAGE; 2) BOLD-fMRI images (TR=1450ms, TE=30ms) acquired when the subjects were under breath hold challenge or under exogenous CO₂ challenge. The breath hold paradigm consisted of 2 consecutive phases (resting and breath holding) repeated 6 times. The resting phase lasted no less than 60 seconds, while the breath holding phase lasted 30 seconds. The challenge lasted 10 minutes. During the exogenous CO₂ challenge, subject wore nose-clip and breathed through a mouth-piece on a MRI-compatible circuit designed to maintain the P_ETCO₂ within ± 1-2 mmHg of target P_ETCO₂.^6,7 The CO₂ challenge paradigm consisted of 2 consecutive phases (normocapnia and mild hypercapnia) repeating 6 times with 3 epochs of 4 mmHg increase and 3 epochs of 8 mmHg increase of P_ETCO₂ above the subject’s resting P_ETCO₂. The normocapnia phase lasted 60-90 seconds, while the mild hypercapnia phase lasted 30 seconds. The total duration of the exogenous CO₂ hypercapnic challenge lasted 10 minutes. Physiological changes including PCO₂, PO₂ and respiration were measured by calibrated gas analyzers and respiratory bellow simultaneously with MRI acquisition. Data analysis: BOLD-fMRI data were imported into the software AFNI⁸ for correction and normalization. Time series of bER during breath hold challenge was derived as the ratio of the change in PO₂ (ΔPO₂ = inspired PO₂ – expired PO₂) to the change in PCO₂ (ΔPCO₂ = expired PCO₂ – inspired PCO₂) measured between end inspiration and end expiration. CVR values were derived from regressing ∆BOLD on bER (CVR_BH-bER), P_ETCO₂ (CVR_BH-PETCO2) and ToB (CVR_BH-ToB) when the subjects were under breath hold challenge. CVR values during exogenous CO₂ challenge were obtained by regressing ∆BOLD on P_ETCO₂ (CVR_CO2-PETCO2). Region-of-interest (ROI) analyses were applied to the CVR values in 160 brain regions parcellated by the software FreeSurfer.⁹ To study the CVR changes in group, one-sample t-tests were used onto the brain volumes with regional CVR_BH-bER, CVR_BH-PETCO2, CVR_BH-ToB and CVR_CO2-PETCO2. Differences were considered significant at false discovery rate adjusted p_fdr<0.05.

Results

Under exogenous CO₂ challenge, most of the brain regions showed increased CVR_CO2-PETCO2 in the subject group especially at the thalamus, insula and putamen (Fig1). For the same group of subjects under breath hold challenge, increased CVR_BH-bER and CVR_BH-ToB were found in most of the brain regions, while no significant changes of CVR_BH-PETCO2 were shown in most brain regions.

Discussion

The interaction between ΔPO₂ and ΔPCO₂ during breath holding is mainly resulting from the systemic metabolic process and different from the effect of exogenous gas administration which is primarily indicated by an increase in ΔPCO₂. During breath holding, changes in systemic PO₂ and PCO₂ may trigger mechanisms which involve the interaction of central and peripheral respiratory chemoreceptors as well as the autonomic system to regulate CBF. bER being better for characterizing dynamic BOLD signal changes under breath hold challenge is closely related to the fact that bER is a ratio which factors out effects of ventilatory volume fluctuations¹⁰ common to both ΔPO₂ and ΔPCO₂. ToB yielded superior correlation result than ΔPCO₂ because ToB indirectly takes into account the duration for both hypoxemia and hypercapnia without being affected by the depth of breathing.

Conclusion

We showed the combined effect of both hypoxia and hypercapnia on cerebral hemodynamic responses measured using BOLD-fMRI. Our findings provide a novel insight on using bER to better quantify CVR changes under breath hold challenge, although the physiological mechanisms of cerebrovascular changes underlying breath hold and exogenous CO₂ challenges are potentially different.

Acknowledgements

This research was carried out in whole at the Athinoula A. Martinos Center for Biomedical Imaging at the Massachusetts General Hospital, using resources provided by the Center for Functional Neuroimaging Technologies, P41EB015896, a P41 Biotechnology Resource Grant supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health, as well as the Shared Instrumentation Grant S10RR023043. This work was also supported, in part, by NIH-K23MH086619.